It is made available under a CC-BY-NC-ND 4.0 International ... · engineered with a nonsense mutation in Pngl at codon 420, resulting in truncation of the C-terminal carbohydrate-binding

TITLE:Defectsintheneuroendocrineaxiscauseglobaldevelopmentdelayina

DrosophilamodelofNGLY1Deficiency

AUTHORS:TamyPortilloRodriguez,JoshuaD.Mast,TomHartl,EthanO.Perlstein*

AFFILIATION:PerlaraPBC,6000ShorelineCourt,Suite204,SouthSanFrancisco,

California94080,UnitedStates

*correspondingauthor:(415)347-8493,[email protected]

MAJORCLASSIFICATION:BiologicalSciences

MINORCLASSIFICATION:Genetics

.CC-BY-NC-ND 4.0 International licenseis made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It. https://doi.org/10.1101/241653doi: bioRxiv preprint

https://doi.org/10.1101/241653

http://creativecommons.org/licenses/by-nc-nd/4.0/

ABSTRACT

N-glycanase1(NGLY1)Deficiencyisararemonogenicmulti-systemdisorderfirst

describedin2014.NGLY1isevolutionarilyconservedinmodelorganisms,including

theDrosophilamelanogasterNGLY1homolog,Pngl.Hereweconductedanatural

historystudyandchemical-modifierscreenonanewflymodelofNGLY1Deficiency

engineeredwithanonsensemutationinPnglatcodon420,resultingintruncation

oftheC-terminalcarbohydrate-bindingPAWdomain.Homozygousmutantanimals

exhibitglobaldevelopmentdelay,pupallethalityandsmallbodysizeasadults.We

developeda96-well-plate,image-based,quantitativeassayofDrosophilalarvalsize

foruseinascreenofthe2,650-memberMicrosourceSpectrumcompoundlibraryof

FDAapproveddrugs,bioactivetoolcompounds,andnaturalproducts.Wefound

thatthecholesterol-derivedecdysteroidmoltinghormone20-hydroxyecdysone

(20E)rescuedtheglobaldevelopmentaldelayinmutanthomozygotes.Targeted

expressionofahumanNGLY1transgenetotissuesinvolvedinecdysteroidogenesis,

e.g.,prothoracicgland,alsorescuesglobaldevelopmentaldelayinmutant

homozygotes.Finally,theproteasomeinhibitorbortezomibisapotentenhancerof

globaldevelopmentaldelayinourflymodel,evidenceofadefectiveproteasome

“bounce-back”responsethatisalsoobservedinnematodeandcellularmodelsof

NGLY1Deficiency.Together,theseresultsdemonstratethetherapeuticrelevanceof

anewflymodelofNGLY1Deficiencyfordrugdiscovery,biomarkerdiscovery,

pharmacodynamicsstudies,andgenemodifierscreens.


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INTRODUCTION

Recessiveloss-of-functionmutationsintheevolutionarilyconservedgeneNGLY1

resultinanultra-raregeneticdiseasecalledNGLY1Deficiency,whichis

characterizedbyglobaldevelopmentaldelay,seizures,smallheadandextremities,

chronicconstipation,lackoftears,andfloppybody(Ennsetal.,2014).NGLY1,short

forN-glycanase1,encodesadeglycosylatingenzymethathydrolyzesN-linked

glycansfromasparagineresiduesofglycoproteins,liberatingoligosaccharidesfor

degradationandrecycling(Suzukietal.,2016).Acomprehensiveclinicalsnapshot

byNationalInstitutesofHealth(NIH)establishedpotentialmeasurableclinical

endpointsandabaselineofdiseaseprogressioninacohortof12patients(Lamet

al.,2017).

ThepathophysiologyofNGLY1Deficiencyhasnotyetbeenfullyresolved.Before

2014,littlewasknownaboutNGLY1function.Itselucidationisthefocusof

numerousresearchgroupsemployingadiversityofdiseasemodels,includingmodel

organismsandhumancells.TwomodelsofthepathogenesisofNGLY1Deficiency

havebeenproposed.

Thefirstmodelisrootedinbiochemistryanddefectsincellularglycoprotein

homeostasis(Huangetal.,2015).NGLY1isanessentialcomponentoftheconserved

proteinqualitycontrolsystemknownasendoplasmic-reticulum-associated


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degradation(ERAD),bridgingp97/VCP-mediatedretrotranslocationofproteins

fromtheERtothecytoplasmforbulkdeglycosylationandsubsequentdegradation

bytheubiquitin-proteasomesystem(UPS)(Suzuki,2015).Theabsenceof

cytoplasmicN-glycanaseactivityhasbeenproposedtoresultininappropriate

cleavageofN-glycansfromglycoproteinsbythedownstreamcytoplasmicenzyme

endo-β-N-acetylglucosaminidase(ENGase),whosenormalsubstrateissoluble

oligosaccharideliberatedbyNGLY1.SuchglycoproteinsmisprocessedbyENGase

wouldretainasingleGlcNacresiduethatmaydestabilizeproteinsandpromote

theiraggregation.SuzukiandcolleaguesobservedevidenceofN-GlcNac

misprocessingandaccumulationinvitroinNGLY1-/-mouseembryonicfibroblasts

(Huangetal.,2015;Fujihiraetal.,2017).BasedonthecollectiveworkofSuzukiand

colleagues,inhibitionofENGasehasbeenproposedasatherapeuticthesisfor

NGLY1Deficiency(Fujihiraetal.,2017;Bietal.,2017).Indeed,aNGLY1-/-ENGase-/-

doublemutantmouseisviablewhileaNGLY1-/-singlemutantdisplaysvarying

degreesoflethalitydependingongeneticbackground(Fujihiraetal.,2017).

However,aNGLY1-/-ENGase-/-doublemutantmouseisnothealthy.Additional

pathogenicmechanismsarerequiredtoexplainNGLY1Deficiency.

Thesecondmodelofpathogenesisisrootedingeneticsanddefectsinthe

deglycosylationofspecificglycoproteinclients,includingbutnotlimitedtothe

masterregulatoroftheconserved26Sproteasome“bounce-back”response,


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NFE2L1(thetranscriptionfactornuclearfactorerythroid2like1alsoknownas

Nrf1)(Radhakrishnan,2010).Nrf1,belongstotheancientbasicleucinezipper

familyoftranscriptionfactorsthatregulatemanydevelopmentalandstress

responsepathwaysinanimals(Kim,2016).TheflyNrf1homolog,cap-n-collar(cnc)

increasestheexpressionofproteasomesubunitgenes,aswellasoxidativeand

redoxstressresponsepathways(Grimberg,2011).Inanunbiasedscreenforgenetic

modifiersoftheproteasomebounce-backresponseinnematodes,Lehrbachand

RuvkundiscoveredthatthenematodehomologofNGLY1,PNG-1,deglycosylatesthe

ER-membraneboundisoformofthenematodehomologofNrf1,SKN-1A.They

furtherdemonstratedthatdeglycosylationofSKN-1AbyPNG-1isrequiredforSKN-

1Atranslocationtothenucleus,andtranscriptionalactivity(Lehrbach,2016).

Inacomplementarystudy,BertozziandcolleaguesrevealedthatNGLY1activityis

requiredforNrf1signalinginmouseembryonicfibroblastsmiceinthesame

mannerthatPNG-1activityisrequiredforSKN-1Afunctioninnematodes(Tomlin,

2017).Infact,theyshowedthatchemicalinhibitionofNGLY1functionpotentiates

cytotoxicitycausedbyproteasomeinhibitioninhumancancercelllines(Tomlin,

2017),whichmirrorstheobservationinnematodesthatpng-1loss-of-function

mutantsareextremelyhypersensitivetoproteasomeinhibitionbybortezomib

(Lehrbach,2016).Jafar-NejadandcolleaguesshowedthattheflyNGLY1/Pnglis

requiredduringembryonicandlarvaldevelopmentinDrosophilaforpost-


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translationalprocessingofDpp,theflyhomologoftheconservedbonemorphogen

protein(BMP)family(Galeone,2017),openingupthepossibilitythatNGLY1is

requiredforthefunctionofmultipleglycoproteinclients.

Herewecarriedoutphenotyping,high-throughputassaydevelopmentanda

chemical-modifierscreenonanewflymodelofNGLY1Deficiency,hereinreferred

toasngly1PL.Of~2,650bioactivecompounds,theecdysteroidinsectmolting

hormone20-hydroxyecdysone(20E)suppressedglobaldevelopmentaldelayin

mutanthomozygotes.ExpressionofahumanNGLY1transgeneintheprothoracic

gland(PG)andsitesofecdysteroidogenesisrescuedglobaldevelopmentaldelayin

mutanthomozygotes.Thesedataindicatethatcellautonomousdefectsinecdysone-

producingorgansandtissuesmayexplaintheglobaldevelopmentaldelayofthe

ngly1PLflies.nglyPLfliesarealsohypersensitivetotheproteasomeinhibitor

bortezomibandtheorganicsolventdimethylsulfoxide(DMSO).Together,these

observationscanbeparsimoniouslyaccommodatedbyamodelwherein

NGLY1/PnglisrequiredforNrf1/cncfunctionintheDrosophilaneuroendocrine

axis.


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METHODS

ngly1PLallelegeneration

Acassettecontainingastopcodonandmw+wasclonedintoamodifiedversionof

pUC57.Homologyarmstothengly1locuswereclonedupstreamanddownstream

ofthecassette.TheguideRNA(GCTGAGGAATAACTTTCGATCGG)wasclonedinto

pCDF3(Portetal.,2014).pCDF3andpUC57withngly1homologyarms,stopcodon,

andmw+wereinjectedintovas-Cas9(Bloomingtonstock#51323).Two

independentmw+F1strainswereestablishedandbalancedstockswerecreated.

Sequencingconfirmedtheintegrationofthestopcodonandmw+(Figure1A)

immediately3’tobp1906699(Release5.57)inchromosome2R(NT_033778.3).

Rateofeclosionandrescuebyhumanngly1transgenestudies

ngly1PL/CyO;actin-switch-gal4;UAS-human-ngly1,w+/+maleswerecrossedto

ngly1PL/CyO,Act-GFPvirginfemales.Thephenotypesofeclosingflieswere

recordedondays9-14afterparentsweremated.Wefoundthattheactin-switch-

gal4transgenicstrainexpressedGal4,despitetheabsenceofRU486.


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Flystrains

Actin5C-switch-gal4(stock#9431)wasobtainedfromtheBloomingtonstock

center.PnglexcisionallelesandUAS-human-ngly1wereobtainedfromDr.Hamed

Jafar-Nejad.2_286,phmandspokGAL4driverswereobtainedfromDr.Michael

O’Connor.

Larvalsizemeasurements

0-4houroldlarvaewereplacedintopetridisheswithstandardflyfoodmediafor3

daysat25°C.Then,larvaewereremovedfromthefood,rinsedinPBS,andplacedin

PBStobeimaged.Theareasofnineteenheterozygousandtwentyhomozygous

larvaeweremeasuredinImageJ.

Platepreparationforchemicalmodifierscreening

100nLcompoundorDMSO(vehicle)wasdispensedfrommotherplatesintowellsof

96welldaughterplatesusingtheEchoacousticdispenser(LabCyte).Then,100uL

ofmoltenstandardflyfoodmedia(molasses,agar,yeast,propionicacid)lacking

cornmeal,butcarrying0.025% bromophenolblue,wasdispensedusingaMultiFlo

(Bio-TekInstruments).Bromophenolbluedyeinthegutsofthelarvaincreases

theirsignaloverbackgroundlaterduringimaging.Plateswereplacedonaplate

shakerandshakenfor1minute,duringwhich,themoltenagarsolidifiedwith


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thoroughlymixedcompound/DMSOineachwell.Plateswerethencoveredwith

adhesivealuminumsealsandstoredat4°Cforuptotwoweeks.

Culturingflylarvaein96-wellplates

Ngly1PL/CyO,Act-GFPflieswereculturedinalargepopulationcage(Genessee)for

uptotwoweeks,wheretheylaideggsongrapejuiceagartrays(Genessee)coated

withathinstripofactiveyeastpaste.Eggcollectionswererestrictedto~6hours

duringmorninghoursandwereplacedinto25°Cfor~24hrs.~0-6hrold1stinstar

larvaewererinsedoffofthetrayswithroomtemperaturewaterandcollectedin

funnel-attached40micronsievestypicallyusedforcellstraining.Toremove

embryocontamination,thelarvae/embryomixturewasincubatedtwotimesin

10.3%inoculationsolution(sodiumchloride,sucrose,10%TritonX-100)forthree

minutes.Mostembryosfloattothetopofthissolutionafterthreeminutes,while

larvaeremaininsuspension.Embryosfromthemixture’ssolutioncanthereforebe

removedwitha10mlpipette.Embryoswerethenaddedtosortingsolution

(Polyethyleneglycol,COPAS200xGPsheathreagent,Tween20)anddrawnintoa

BioSorter(UnionBiometrica)forsortinganddispensinginto96well

compound/mediabearingplates.ThreeGFPnegativehomozygotesweregated

fromheterozygotesanddispensedintothe11leftmostcolumnsoftheplate,and

wellsG12andH12.ThreeGFPpositiveheterozygotesweredispensedintowells

A12,B12,C12,D12,E12,andF12.Plateswerethencoveredwithpermeable


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adhesivesealsandincubatedat25°Cforthreedays.Approximately18plateswere

dispensedintoperday,andlarvalviabilitywasnotaffectedbytheirtime

submergedindispensingsolution(<4hours).

Preparationofplatesforimaging

Ontheterminaldayoftheassay(day3),gaspermeablesealswereremovedand

plateswerefilledwith20%sucrosesolutionmadeacidicwithhydrochloricacid(pH

2)andcarryingdefoamer(FiveStarDefoamer105-2oz).Plateswerethenvortexed

andmoresolutionwasaddedtosuspendlarvaetoafixedfocalplane.Plateswere

thenimaged.

Imageanddataanalysis

TheFlyImagerusesaSonya7riicamera,controlledoverUSBbythegphoto

softwaretogeneratefullplateimagesthatarethenrunthroughalarvaldetection

algorithm.Thealgorithmfirstbuildsanimageofwhatthewellwouldlooklikewhen

itisempty.Todothisitexcludesareasnearedges(sincethoseareprobablylarvae)

andinterpolatesacrossthegaps.Itthenlooksatthedifferencebetweentheimage

andestimatedemptybackground,selectingareaswithahighdifferencetobelarvae.


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20Efeedingandeffectondevelopmentaltiming

20E(Enzo)wasdissolvedin100%ethanolandaddedtomoltenstandardflyfood

mediaat200µMandaddedtovials.Anequivalentsetofvialswithanequalamount

ofethanolwasaddedfornegativecontrols.0-6hourlarvaeweredispensedwith

theBioSorterintothevialsandincubatedat25°Cfor12days.Thenumberoflarvae

thatpupariatedwererecordedonkeydaysat8am,12pm,and4pm,andthenumber

ofadultsthateclosedwererecordedondays9–12atasingletime.

Rescue of developmental delay with ring and prothoracic gland expression of human

ngly1

Ringglandexpressionofhumanngly1wasdrivenusingtheGAL4/UASsystem

(BrandandPerrison,1993)andringglanddrivers286-GAL4,spok-GAL4,andphm-

GAL4inngly1PL/ex20compoundheterozygotes.Adultswereallowedtolayeggsfor8-

16hours,andthegenotypesforalleclosingflieswererecorded.Thenumberofring

glandrescuedflieseclosedwasnormalizedtothenumberofeclosedsibling

homozygotesnotcarryingthedriver.


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RESULTS

Generationofthengly1PLflyandcharacterizationofitsphenotypes

WeusedCRISPR/Cas9tocreateanewalleleofPnglwithaprematurestopcodon

andaselectablemarkerinsertedupstreamofthePAWdomain,hereinreferredto

simplyas“ngly1PL“(Fig.1A).Webenchmarkedngly1PLagainstthepreviously

describedPnglgeneticnullmutantgeneratedbyP-elementexcision(Pnglex20),

whichcausesdevelopmentaldefectsandsemi-lethalitywithfewadultescapers

(Funakoshi,2010).ngly1PLhomozygotesaredelayedinthelarval-to-pupal

transitionbyoneday,anddelayedtoeclosionby2days(Fig.1B).Asthree-day-old

larvae,ngly1PLhomozygotesare~75%thesizeoftheirheterozygotesiblings(P<

1X10-10,student’st-test)(Fig.1D).Consistentwiththesemi-lethalityofPnglexcision

alleles,wealsoobservedthatthengly1PLhomozygotemutantexhibitscold-sensitive

pupallethality,meaningnosurvivalbelow18˚C(datanotshown).Timetoeclosion

inngly1PLhomozygotesiscompletelyrescuedbyubiquitousexpressionofahuman

NGLY1(hNGLY1)transgene(Fig.1B).Thesmallbodysizephenotypeofngly1PL

homozygoteadultsisalsorescuedbyubiquitousexpressionofhNGLY1(Fig.1C).

Theseresultswhenfliesarerearedinstandardvialssuggestedphenotypessuitable

forhigh-throughput,whole-organismphenotypicscreeningateachstageoffly

developmentfrom1stinstarlarvaeonwards.


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Wedecidedtooptimizeahigh-throughputlarvalsizeassayin96-wellplatesfor

severalreasons.Assayminiaturizationfrom30mLvialsto96-wellplatesinvolves

reducingthenumberofanimalstestedbyafactorof10,e.g.,30animalspervial

versus3animalsperwell.Thelarvalsizedifferencebetweenngly1PLheterozygote

larvaeversusngly1PLhomozygotelarvaewasmorestatisticallyrobustin96-well

platesthanthetimingtopupationdifferenceorthetimingtoeclosiondifference.A

3-dayassayversusa7-11dayassayallowedforfasteroptimizationcycletimes.

Timetopupationandtimetoeclosionwouldbesecondaryassaysinvialformatto

ensureprimaryscreeninghitsrescueglobaldevelopmentaldelay,notjust

developmentaldelayinlarvae.

Asapreludetodrugscreening,weestablishedthetolerabilityofngly1PL

homozygotelarvaetodimethylsulfoxide(DMSO),theorganicsolventforalmost

everycompoundlibrary,includingtheMicrosourceSpectrumcollection.The

maximumtolerateddoseofDMSO,therefore,setsaceilingonthefinalassay

screeningconcentration.Unexpectedly,weobservedthatngly1PLhomozygotesare

extremelyhypersensitivetoDMSOcomparedtotheheterozygotecontrol(Figure

2).Weestimatedamaximumtolerateddoseinthengly1PLhomozygoteof0.1%v/v

DMSO(14mM)(Fig.2A,C),whichisactuallyseveralfoldmoresensitivethanPngl

excisionallele20(Fig.2B).Incomparison,wild-typeDrosophilaexhibitadult

lethalitystartingat1%v/vDMSO,larvallethalityrangingfrom2%v/vto3%v/v


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DMSO,anda“noobservedadverseeffectlevel”(NOAEL)doseof0.3%v/vDMSO

(Nazir,2003).DMSOhypersensitivityappearstobespecifictothengly1PL

homozygoteinalimitedcomparisontoothermutants(datanotshown).Forthe

purposesofdrugscreening,weexploitedDMSOasasensitizerinthelarvalsize

assay.Exposureofmutantlarvaeto0.1%v/vDMSO,whichwouldentailafinal

assayscreeningconcentrationof10µMforeachlibrarycompound,waspotent

enoughtoincreasethedynamicrangeandimprovetherobustnessoftheassay

whilesparinglarvallethality.

Astatisticallyrobusthigh-throughputimage-basedlarvalsizechemicalmodifierpilot

screenyieldedonevalidatedhit,20-hydroxyecdysone(20E)

Wepositedthatthesmalllarvalsizephenotypeofngly1PLhomozygotemutants

couldbeusedtodiscoversmall-moleculesuppressorsthatprovideinsightintothe

pathophysiologyofNGLY1/Pngldeficiencyinthefly.Tothatend,wedevelopeda

novelimage-basedassaytocultureDrosophilalarvaeinclear-bottom96-wellplates

whereeachwelleithercontainsauniquesmallmoleculeorvehicle.Usinga96-well-

plateformatallowedustotakeadvantageofexistinglabautomationformanaging

multi-wellplatesinhigh-throughputscreening(HTS)applications,includinga

whole-organismsorteranddispenser.Ourmethodincludesstepstodissolveand

dilutepre-existingflyfoodsothatlarvaecanbefloatedtoafixedfocalplaneand


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thenimagedwithcustomplateimager.Softwarewaswrittentomeasuretheoverall

areaoffloatedlarvaetoenabletheidentificationofsmallmoleculesthat

significantlyincreasethesizeofngly1PLhomozygotelarvaevsvehicle-fedlarvae.

WeselectedtheMicrosourceSpectrumcollectionforapilotscreen.Thelibrary

contains2,532uniquecompoundsincluding~600FDAapproveddrugs,~800

compoundsthathavereachedclinicaltrialstagesintheUSA,~400drugsthathave

beenmarketedinEuropeorJapanbutnottheUSA,~600bioactivetoolcompounds,

and~800naturalproducts.Threelarvaeperwellwereculturedin96-wellplates

withcontrolwellscomprisingthetwooutermostcolumns.Weusedngly1PL

homozygouslarvaefedvehicle(0.1%v/vDMSO)asanegativecontrol.Weinclude

twopositivecontrolgroups:thefirstgroupconsistofngly1PLheterozygouslarvae

fedvehicle,andthesecondgroupconsistsofngly1PLhomozygouslarvaecultured

withoutDMSO.Theremaining80wellscontainedngly1PLhomozygouslarvaefeda

uniquelibrarycompoundat10µMplus0.1%v/vDMSO.Animageofa

representativedrugscreeningplatewithzoomed-inreferencewellsisshownin

Figure3.

Weperformedthescreenintriplicate,meaningthreeindependentbiological

replicates.Therewasarobuststatisticalseparationbetweenpositiveandnegative

controls(Fig.4A).Onaverage,ngly1PLhomozygoteswerehalfthesizeofngly1PL


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heterozygotes,althoughsomevariationinsizewasobservedineachgenotype.75%

ofallscreeningplates(73of96)hadaZ’factor>0;mostofthescreeningplates

withhighvariabilitybelongedtothesecondreplicate(Fig.4B).Aweakpositive

correlationexistedinpairwisecomparisonsofeachbiologicalreplicatewhenall

datapointsareincluded(Fig.5A).WhenweonlyincludedwellswithZscoresless

than-2orgreaterthan2,thepositivecorrelationincreasedsignificantlyonaverage

(Fig.5B).Forexample,inthepairwisecomparisonofreplicate1versusreplicate2,

thecorrelationimprovesfromR2=0.05977inthefulldatasettoR2=0.48171inthe

reduceddataset(withpositiveandnegativecontrolsremovedaswell).

Weinitiallyconsidered162pre-hitswithaZscoreof>2intwoofthreebiological

replicatesasprimaryscreeningpositives(Table1).Overtwo-thirdsofthosepre-

hitsprovedtobefalsepositivesforoneoftwoofthefollowingreasons.First,

uneatenflyfoodinthewelloccasionallyincreasedtheimagebackgroundartificially

inflatingthecalculatedareaofthelarvae.Second,becausePnglmutantsare

hypersensitivetoDMSO,anyfailureincompounddispensingorvariabilityinDMSO

levelsduetohydrationresultedinlargerlarvae.Forty-fivecompoundswithaZ-

scoreof>2intwoofthreereplicateswereconsideredfurtherbecausetheirwells

didnothaveobvioushighbackgroundorlow/noDMSO.Therawimagesofthewells

containingthose45compoundsweremanuallyinspected,and22hadaclear

differencefromcontrols.Ultimately,22compoundswerefoundtohaveaZof>2in


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twoofthreereplicatesanduponmanualinspection,thewellswiththose

compoundsappearedtohavelarvaelargerthanthenegativecontrolsinthesame

plate.Twoofthose22compoundshadaZof>2inthreeofthreereplicates,were

countedtwiceinthiscomparison,andthusthefinalsetofpre-hitswetestedfurther

consistsof18uniquecompounds.

All18ofthepre-hitsfromthescreenwereordered,dissolved,andretestedin

attempttoreproducerescueinvialformatwithlargernumbersofanimals(Fig.5C,

Table2).Onecompound,20-hydroxyecdysone(20E),rescuedthedevelopmental

delayofngly1PLhomozygotelarvaedevelopmenttopupae(Fig.6B),buthadno

effectondevelopmentaltimingofngly1PLheterozygotelarvae(Fig.6A).Moreover,

thesuppressiveeffectof20Epersistedtoadulthood,resultinginastatistically

significant4-foldincreaseineclosionpercentage(Fig.6E).Asacontroltoruleouta

simpleecdysonebiosynthesisdefect,weshowedthatthe20Eprecursor7-

hydroxycholesterol(7D)failedtorescuengly1PLhomozygotelarvaedevelopmentto

pupae(Fig.6D).Ifsynthesisof20Eisfaulty,itislikelyatapointdownstreamof7D.

Wecouldnotreproduciblyvalidateanyoftheother17pre-hits,sowefocusedour

effortsonunderstandingthemechanism-of-action(MoA)of20E.Therefore,this

pilotscreenhadanextremelylowhitrateof0.04%(1/2532).


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20Eimplicatestheflyneuroendocrineaxisasparticularlysensitivetolossof

NGLY1/Pnglfunction

20EdrivesmetamorphosisinDrosophilaandarthropodsgenerally(Faunes,2016).

Dietarycholesterolformsthebasisof20E,andall20Eprecursorsaresynthesizedin

theprothoracicgland,anorganof<50cellsthatcomprisespartofthelargerring

gland.Theimmediate20Eprecursor,ecdysoneor“E”,ispackagedintosecretory

vesicles,secreted,anddistributedbythehemolymphthroughouttheanimals.Eis

convertedto20Eintheseperipheraltissues,andinitiatessignalingcascadesand

geneexpressioninducingphysiological,morphological,andbehavioralchangeswith

eachmolt,ordevelopmentaltransition.

Totestwhetherthe20EinsufficiencyinPngl-deficientmutantsisautonomousto

theringgland,weexpressedaUAS-drivenhNGLY1transgenethatcanrescueglobal

developmentaldelaywhenexpressedubiquitously(Fig.1B,C),intheringgland

withthe2-286-GAL4driver.Tocontrolforoff-targetmutationsduetostrain

backgroundconfoundingourresults,wetestedthetrans-heterozygouscombination

ofPnglalleles,ngly1PL/ex20.Mostngly1PL/ex20larvaenotexpressingthehuman

ngly1transgeneeclosedonDay10.Incontrast,mostngly1PL/ex20larvaeexpressing

humanNGLY1intheringglandpupatedonDay9(Fig.7A).Asidefromthering

gland,2-286-GAL4alsodrivesexpressioninthesalivarygland,fatbody,andcuticle


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inlarvae(Timmonsetal.,1997).Weobservedasimilarrescueeffectwhenhuman

ngly1transgeneexpressionisdrivenbytworing-gland-specificdrivers,phantom-

GAL4(phm)(Fig.7B)andspookier-GAL4(spok)(Fig.7C).phmandspokencode

cytochromeP450monooxygenasesintheecdysonebiosyntheticpathway(Rewitz,

2007).Together,thesedataindicatethatNGLY1/Pnglisnecessaryfornormal

functionoftheringglandtoenableproperlevelsof20Etocirculatethroughoutthe

developinganimalanddrivedevelopmentaltransitions.

NGLY1/Pngldeficientfliesarehypersensitiveproteasomeinhibition

Asmentionedabove,lossofNGLY1sensitizesnematodesandhumancancercell

linestoproteasomeinhibition.Wepredictedthatngly1PLhomozygotemutants

wouldexhibithypersensitivitytobortezomib.Indeed,bortezomibcaused100%

lethalityofngly1PLhomozygouslarvaeat5µM,whilelethalitywasnotobservedin

ngly1PLheterozygouslarvaeuntiladoseof25µM(Figure8.).Inaddition,thesizeof

ngly1PLhomozygousmutantswasreducedby<50%whentreatedwith1µM

bortezomib,andto~50%ngly1PLheterozygouslarvaeat~10µMbortezomib.

Thesedataindicatethatthehalf-maximalinhibitoryconcentration(IC50)of

bortezomibtoreducelarvalgrowthis~10Xlessinngly1PLhomozygote.


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DISCUSSION

WesuccessfullygeneratedanewDrosophilamodelofNGLY1Deficiency,optimized

ahigh-throughputwhole-animalphenotypicassayoflarvalsize,andthen

demonstratedtherapeuticrelevanceinaproof-of-conceptdrugrepurposingscreen.

While20EitselfshouldnotbeconsideredadrugcandidateforNGLY1Deficiencyin

humans,thefactthatitisachemicalsuppressorimplicatestheneuroendocrineaxis

inthepathophysiologyofPngldeficiencyinflies.Inotherwords,eventhough20Eis

aninsect-specificdevelopmentalhormone,theneuroendocrineaxisandsteroid-

deriveddevelopmentalhormonesareconservedinmammalsandmayplayarolein

NGLY1Deficiencyinhumans.Collectively,ourfindings–moststrikingly,

hypersensitivityofthengly1PLmutantbothtobortezomibandtoDMSO–alignwith

resultsobservedinnematodes(LehrbackandRuvkun,2017)andhumancells

(Tomlin,2017)thatpointtotheessentialandconservedroleofNGLY1inregulating

thefunctionofglycoproteinclients,specificallyNrf1andtheproteasomebounce-

backresponse.

Infact,wecanalreadyproposeamechanismtolinkNrf1functionto

ecdysteroidogenesisandtheneuroendocrineaxisinflies.TheflyhomologofNrf1is

thelongestisoformofcnc,CncC,whichcontainsaconservedN-terminalleucinerich

transmembraneregiontargetingCncCtotheER(Grimberg,2011).Specificlossof


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CncCintheprothoracicglandreducestheexpressionofecdysonebiosyntheticgenes

andresultsindelayedtimingtopupation(DengandKerppola,2013).RNA

interferenceoftheColoradopotatobeetlehomologofCncCalsoreduced

ecdysteroidogenesispathwaygeneexpressionanddelayedtimingpupation,which

couldberescuedby20Esupplementation(Sunetal.,2017).Ourdatamakeastrong

testableprediction:CncCisthefunctionalequivalentofSKN-1Ainnematodesand

Nrf1inmammals,andthereforeisasubstratefordeglycosylationbyPnglinflies.A

secondtestablepredictionisthattheflyhomologofnematodeDDI-1andhuman

DDI2,ringslost(ringo),actsdownstreamofPngltoproteolyzeCncC,generatinga

mature,nuclear-activespecies.

Thereareotherexplanationsforhow20Emightrescueglobaldevelopmentaldelay

inthengly1PLmutantthatdonotdirectlyinvolveNrf1/CncC,orthatmayactin

concertwithlossofNrf1/CncCactivity.First,ngly1PLmutantsmaynotpackage

ecdysoneintosecretoryvesiclesproperly,ormaybedefectiveinsecreting

ecdysone.Second,signaltransductionmediatedbytheinteractionbetween20Eand

theecdysonereceptor(EcR)mightnotbefullyoperational,andsoextra20Eboosts

thisflawedsignaling.Third,ecdysonesecretionbytheringglandisinducedbytwo

neuronsthatsynapseontotheprothoracicglandandsecretetheneuropeptide

prothoracicotropichormone,orPTTH.PTTHcontactsthereceptortyrosinekinase

Torsotoinitiatesignalingleadingtoecdysonesecretion.Perhapsthereisaflawin


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PTTHsecretionorTorsosignaling.Fourth,damagetodevelopinglarvaltissues,or

starvation,mayimpingeon20Etofinetuneorganismdevelopmentsothata

properlyproportionedandnourishedanimalcandevelopfullytoadulthood.The

ngly1PLmutantmayhavedamagedtissues,forexamplethroughprotein-aggregate

toxicityorproteasomestress;oritmayhavesomedegreeofstarvation,forexample

ifthegutcannotattainnutrientsproperly.20Efeedingmaybypassdelaysinduced

bythishypotheticaltissuedamage/starvation.

ACKNOWLEDGMENTS

WethanktheGraceScienceFoundationforfundingthiswork.WethankDr.Kevin

Leeforplanningandexperimentplanning.Drs.HamedJafar-NejadandTadashi

Suzukikindlyprovidedreagents.PeterSanddesignedandbuiltourcustomfly

imagerandanalysissoftware.Dr.TomLeeprovidedassistanceduringngly1PL

mutantstrainconstruction.Dr.RebeccaChoyprovidedearlyguidanceonhowto

scaleupflyhusbandry/culturing.


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Figure1.Genotypingandphenotypingngly1PLallele.A)WeusedCRISPRtocreateanalleleofngly1withastopcodonandwhite+transgeneinsertedupstreamofthePAWdomain.Sequencingconfirmedtheintegrationofthestopcodonandmw+immediately3’tobp1906699(Release5.57)inchromosome2R(NT_033778.3).B)Ubiquitousexpressionofahumanngly1transgenerescuedthe2-daydevelopmentdelaytoeclosionphenotypeobservedonngly1PLhomozygotes.C)ngly1PLhomozygousadultfliesaresmallerthantheirhomozygoussiblingsexpressingthehumanngly1transgene.D)ngly1PLhomozygousaredevelopmentallydelayed,at3daysold,thelarvaeare25%smallerthantheirheterozygoussiblings(p<0.01).


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Figure2.ngly1PLhomozygotesarehypersensitivetodimethylsulfoxide(DMSO).A)Animageofngly1PLhomozygouslarvaegrownin96-wellplateformatonfooddosedwithdifferentconcentrations(0%-0.4%)ofDMSO.ngly1PLhomozygouslarvaearehypersensitivetoDMSO,adecreaseinlarvalsizeisobservedstartingat0.025%DMSOandlarvalsizeisdecreaseasthedoseofDMSOisincreased.ThetimetopupationofB)ngly1Ex20orC)ngly1PLhomozygouslarvaerearedonfoodtreatedwithDMSO.HomozygousmutantlarvaeexhibithypersensitivitytoDMSOshowingadelayedtimetopupariateandincreasedlethality.


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Figure3.Layoutofanngly1assayplatewithexamplesofcontrolsandpre-hitcompound.Larvaewereculturedin96-wellplateswithnegativecontrolsontheleftmostwells(column1)whichcarriedngly1PLhomozygouswithvehicleDMSOat0.1%.80testingwellswereinthemiddle(columns2-11)whichcarriedngly1PLhomozygouslarvaewithacompoundat10uM,andDMSOat0.1%.Rightmosttopwells(12A-12F)containpositivecontrolgroup1withngly1PLheterozygouslarvaeand0.1%DMSO.Rightmostbottomwells(12G-12H)containpositivecontrolgroup2withngly1PLhomozygouslarvaewithoutDMSO.ThecompoundinwellG4wasconsideredapre-hitcompound.


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Figure4.Theassayhadaconsistentdifferencebetweenpositiveandnegativecontrols.A)AconsistentseparationbetweenpositiveandnegativecontrolswithZ’Factor>0,indicatedarobustassaythatcouldidentifychemicalsuppressors.B)Threereplicatesofa32platelibrarywereanalyzedand73of96plateshadaZ’Factor>0whichfurthersuggestarobustassaytoidentifychemicalsuppressors.


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Figure5.Positivecorrelationbetween3Xreplicates.A)ThethreepairwisecomparisonsofZ-scoresshowpositivecorrelationsindicatingthatasetofsmallmoleculescouldmodifythesmalllarvalsizephenotypeB)ThepositivecorrelationsbetweenreplicatesismoreevidentwhenonlyplottingZ-scoresof<-2or>2.C)Larvalsizeisrescuedwhen20Eisfedtongly1PLhomozygouslarvae.


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Figure6.20-hydroxyecdysone(20E),butnotanearlierecdysonepathwayprecursor(7-dehydrocholesterol),partiallyrescuesdevelopmentaldelayandlethalityinngly1PLmutants.ThetimetopupationofA)ngly1PLheterozygousorB)ngly1PLhomozygouslarvaerearedonfoodtreatedwithdifferentconcentrationof20E.20Edidnotimpactdevelopmentalrateofheterozygousngly1PLlarvaetopupation,butrescueddevelopmentdelayofhomozygousngly1PLlarvaetopupationat200µM.ThetimetopupationofC)ngly1PLheterozygousorD)ngly1PLhomozygouslarvaerearedonfoodtreatedwithdifferentconcentrationof7-d.7-ddidnotimpactdevelopmentalrateofheterozygousorhomozygousngly1PLlarvaetopupation.E)Thefractionofanimalssurvivingtoeclosion.20E,butnot7-d,rescuedlarvallethalityofngly1PLhomozygousat200uM.


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Figure7:ngly1isnecessaryfornormalfunctionoftheringgland.Thefractionofngly1PL/ngly1Ex20compoundheterozygoteswithhumanngly1drivenbytheringglanddriver(blue)A)2_286-GAL4B)spokier-GAL4,orC)phantam-GAL4survivingtoeclosioncomparedtosiblingcontrolslackingadriver(red).A)Compoundheterozygousngly1PL/ngly1Ex20larvaeexpressing2_286>ngly1eclosedearlierandhadlowerlethalitythancontrolflies.Compoundheterozygousngly1PL/ngly1Ex20larvaeexpressingspokier>ngly1orphantam>ngly1eclosedlowerlethalitythancontrolflies.


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Figure8.NGLY1larvaeare≥10Xmoresensitivetotheproteasomeinhibitorbortezomib.Imagesof3dayngly1PL+/-heterozygousoragematchedngly1PL-/-mutantlarvaeraisedonfoodtreatedwith0,1,5,10or25µMbortezomib.Bortezomibdelayslarvaldevelopmentalprogressioninngly1PLhomozygouslarvaemoreseverelythanngly1PLheterozygouslarvaeleadingtosmallersizedlarvae,andislethalforhomozygotesatconcentrationsequaltoorgreaterthan5µM.


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Table 1. Whittling screen pre-hits to a set of promising compounds to test in validation studies. Ultimately, 22 compounds were found to have a Z of > 2 in 2 of 3 replicates and upon manual inspection, the wells with those cpds appeared to have larvae larger then the negative controls. Two of those 22 compounds had a Z of > 2 in 3 of 3 replicates, were counted twice in this comparison, and thus the final set to consider further were 18 unique compounds.


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It is made available under a CC-BY-NC-ND 4.0 International ... · engineered with a nonsense mutation in Pngl at codon 420, resulting in truncation of the C-terminal carbohydrate-binding